Overexpression, secretion and antifungal activity of the Saccharomyces cerevisiae chitinase
The excessive use of pesticides to prevent the outbreak of plant diseases holds various disadvantages for the cost-effectiveness of agricultural production practices, the quality of the end products, the environment and the labour force. The mounting public resistance to the use of hazardous pesticides on agricultural crops has hastened the search for natural antimicrobial peptides, enzymes and other types of biological control agents. The objective of this study was to investigate the potential of the antifungal activity of a yeast-derived chitinase for possible future agricultural applications. When plants are exposed to fungal pathogens, they produce pathogenesis-related (PR) defence proteins, such as chitinases, in order to degrade the chitin in the cell walls of the attacking fungi, thereby inhibiting further fungal growth and the development of hyphae. We have cloned the Saccharomyces cerevisiae chitinase gene (CTS1-2) into a multicopy 2μ-based plasmid and overexpressed it under the control of the phosphoglycerate kinase I gene (PGK1) promoter (PGK1P) and terminator (PGK1T) sequences. Secretion of the recombinant CTS1-2-encoded chitinases was directed by the native leader peptide, or by the secretion signals of either the yeast mating pheromone α-factor (MFα1S) or the Trichoderma reesei β-xylanase 2 (XYN2S). Northern blot analysis confirmed the PGK1PT-directed expression of CTS1-2 and an indirect enzyme assay was used to compare the efficiency of secretion of the three different recombinant chitinases. These recombinant chitinases were also shown to effectively inhibit spore germination and hyphal growth of Botrytis cinerea, an economically important fungal pathogen of several agricultural crop plants, including grapevine. This study could lead to the development of yeast chitinases and/or chitinolytic yeasts as biocontrol agents to combat plant diseases and reduce the use of chemical pesticides in the agricultural industry.